Basics of Centrifugation

Reprinted with permission of THERMO

The purpose of this tutorial is to introduce basic concepts of centrifugation, including vocabulary, centrifuge
and rotor types, separation techniques, and even gradient selection. For further details regarding
centrifugation, please refer to the Sorvall® and Heraeus recommended reading the literature cited in the
reference section.

Contents.

I. Introduction
II. Increasing the effect of gravity: the centrifuge
III. Types of centrifugal separation
IV. Rotor categories
V. Selection of centrifuge tubes
VI. Common centrifuge vocabulary and formulas
VII. Reference and suggested readings
I. Introduction.
Centrifugation is one of the most important and widely applied research techniques in biochemistry, cellular
and molecular biology, and in medicine. Current research and clinical applications rely on isolation of cells,
subcellular organelles, and macromolecules, often in high yields.

A centrifuge uses centrifugal force (g-force) to isolate suspended particles from their surrounding medium on
either a batch or a continuous-flow basis. Applications for centrifugation are many and may include
sedimentation of cells and viruses, separation of subcellular organelles, and isolation of macromolecules
such as DNA, RNA, proteins, or lipids.

II. Increasing the effect of gravity: the centrifuge.

Many particles or cells in a liquid suspension, given time, will eventually settle at the bottom of a container
due to gravity (1 x g). However, the length of time required for such separations is impractical. Other
particles, extremely small in size, will not separate at all in solution, unless subjected to high centrifugal
force. When a suspension is rotated at a certain speed or revolutions per minute (RPM), centrifugal force
causes the particles to move radially away from the axis of rotation. The force on the particles (compared to
gravity) is called Relative Centrifugal Force (RCF). For example, an RCF of 500 x g indicates that the
centrifugal force applied is 500 times greater than Earth’s gravitational force. Table 1 illustrates common
centrifuge classes and their applications.

Table 1. Classes of centrifuges and their applications.

III. Types of Centrifugal Separations.
1. Differential centrifugation. Separation is achieved primarily based on the size of the particles in
differential centrifugation. This type of separation is commonly used in simple pelleting and in obtaining
partially-pure preparation of subcellular organelles and macromolecules. For the study of subcellular
organelles, tissue or cells are first disrupted to release their internal contents. This crude disrupted cell
mixture is referred to as a homogenate. During centrifugation of a cell homogenate, larger particles
sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by
differential centrifugation. A cell homogenate can be centrifuged at a series of progressively higher g-forces
and times to generate pellets of partially-purified organelles.

When a cell homogenate is centrifuged at 1000 x g for 10 minutes, unbroken cells and heavy nuclei pellet to
the bottom of the tube. The supernatant can be further centrifuged at 10,000 x g for 20 minutes to pellet
subcellular organelles of intermediate velocities such as mitochondria, lysosomes, and microbodies. Some of
these sedimenting organelles can obtained in partial purity and are typically contaminated with other
particles. Repeated washing of the pellets by resuspending in isotonic solvents and re-pelleting may result in
removal of contaminants that are smaller in size (Figure 1). Obtaining partially-purified organelles by
differential centrifugation serves as the preliminary step for further purification using other types of
centrifugal separation (density gradient separation).

2. Density gradient centrifugation. Density gradient centrifugation is the preferred method to purify
subcellular organelles and macromolecules. Density gradients can be generated by placing layer after layer
of gradient media (Table 2) such as sucrose in a tube with the heaviest layer at the bottom and the lightest
at the top in either a discontinuous or continuous mode. The cell fraction to be separated is placed on top of
the layer and centrifuged. Density gradient separation can be classified into two categories. 2a. Rate-zonal
(size) separation. 2b. Isopycnic (density) separation.

2a. Rate zonal (size) separation

Rate-zonal separation takes advantage of particle size and mass instead of particle density for
sedimentation. Figure 2 illustrates a rate-zonal separation process and the criteria for successful rate-zonal
separation. Examples of common applications include separation of cellular organelles such as endosomes or
separation of proteins, such as antibodies. For instance, Antibody classes all have very similar densities, but
different masses. Thus, separation based on mass will separate the different classes, whereas separation
based on density will not be able to resolve these antibody classes.

Certain types of rotors are more applicable for this type of separation than others. Please See rotor
categories (below) and Table 2.